Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

ABSTRACT

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

TECHNICAL FIELD

The present invention relates to a novel coenzyme-linked glucosedehydrogenase (hereinafter, may be referred to as “GLD”), apolynucleotide encoding the same, a method for producing the same, amethod for producing the GLD, and use of the GLD.

BACKGROUND ART

The glucose content in blood is considered an important marker fordiabetes. A diagnosis of diabetes is made by a simplified measurement(Point-of-Care Testing: POCT) such as a simplified test conducted byclinical staff or the like, or a self-inspection conducted by a patient,in addition to a clinical examination conducted in a hospitalexamination room or the like.

Although the simplified measurement is conducted using a glucosediagnostic kit or a measurement apparatus such as a biosensor or thelike (POCT apparatus), a glucose oxidase is conventionally used in thePOCT apparatus. However, the glucose oxidase depends on dissolved oxygenconcentration, and thereby errors occur in measured values. Accordingly,use of glucose dehydrogenase which is not influenced by oxygen isrecommended.

There are, as the glucose dehydrogenase, NAD coenzyme-unlinked glucosedehydrogenases of which the coenzyme is nicotinamide adeninedinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP)and coenzyme-linked glucose dehydrogenases of which the coenzyme ispyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), orthe like. Among them, the coenzyme-linked glucose dehydrogenases areadvantageous in that they are less liable to be affected bycontamination components in comparison with the NAD coenzyme-unlinkedglucose dehydrogenases, and they realize high measurement sensitivityand production of the POCT apparatuses at low cost.

However, conventional pyrroloquinoline quinone (PQQ)-type glucosedehydrogenases are disadvantageous in that the stability thereof is low,and they easily react with maltose or galactose. Maltose is a sugar usedin a transfusion. When the PQQ-type glucose dehydrogenases react withmaltose, the POCT apparatus which measures blood sugar levels indicatesa higher blood sugar level than an actual blood sugar level. As aresult, the patient injects an excessive amount of insulin, and therebysuffers from hypoglycaemia, which causes consciousness disorder or acomatose state, which has attracted tremendous interest.

In particular, the blood sugar POCT apparatus is used to measure theblood sugar level and the importance thereof has increased due to itsconvenience in patient self-care and medication, and thus self bloodsugar monitoring apparatuses (Self-Monitoring of Blood Glucose: SMB)have been increasingly used in the home. Accordingly, the demand forrealizing measurement accuracy is deemed to be very high.

In actuality, a notification calling for attention with respect to useof blood sugar testing apparatuses with an enzyme that utilizes PQQ as acoenzyme was issued to patients receiving maltose transfusion from theJapanese Ministry of Health, Labor and Welfare on February, 2005 (Feb.7, 2005; Pharmaceutical and Food Safety Notification No. 0207005, andthe like).

On the other hand, there have been reported, as the coenzyme-linkedglucose dehydrogenases which catalyze dehydrogenation of glucose withFAD as the coenzyme, enzymes originating from Agrobacterium tumefaciens(J. Biol. Chem. (1967) 242: 3665-3672), enzymes originating fromCytophaga marinoflava (Appl. Biochem. Biotechnol. (1996) 56: 301-310),enzymes originating from Halomonas sp. α-15 (Enzyme Microb. Technol.(1998) 22: 269-274), enzymes originating from Agaricus bisporus (Arch.Microbiol. (1997) 167: 119-125, Appl. Microbiol. Biotechnol. (1999) 51:58-64), and enzymes originating from Macrolepiota rhacodes (Arch.Microbiol. (2001) 176: 178-186). These enzymes oxidize a hydroxyl groupat the 2-position and/or 3-position of glucose, and exhibit a highactivity toward maltose, but low selectivity to glucose. Althoughcoenzyme-linked glucose dehydrogenases originating from Burkhorderiacepacia with a high activity toward maltose are also known, theirnatural type enzyme is a heterooligomer enzyme composed of threesubunits α, β, and γ, and known as a membrane-binding enzyme.Accordingly, there are disadvantages in that solubilization treatment isrequired to obtain the enzyme, and cloning of necessary subunits issimultaneously required to realize sufficient activity by cloning.

In the Society for Biotechnology, Japan (October 28 to 30, 2002), therewas a presentation regarding the substrate selectivity (activity againstmaltose and activity against galactose, with respect to the activityagainst glucose which is assumed to be 100%) in which SM4 strainexhibited 40% and 105%, JCM5506 strain exhibited 43% and 132%, JCM550strain exhibited 57% and 123%, JCM2800 strain exhibited 83% and 108%,JCM2801 strain exhibited 74% and 117%, IFO14595 strain exhibited 38% and104%, and IFO15124 strain exhibited 74% and 148%, and the presenterthereof stated that these strains exhibited high activity againstmaltose, which was disadvantageous if used for a self blood sugarmonitoring apparatus, and therefore the presenter was going to improvethe substrate selectivity by changing the sequence thereof.

In contrast, inventors of the present invention invented a novel solublecoenzyme-linked glucose dehydrogenase of which the coenzyme is FAD andwhich is not a membrane-bound type, and filed a patent application(Patent Document 1). The coenzyme-linked glucose dehydrogenase disclosedin Patent Document 1 oxidizes a hydroxyl group at the 1-position ofglucose, is excellent in substrate-recognizing ability against glucose,is not influenced by dissolved oxygen, and exhibits low activity towardmaltose (activity against maltose of 5% or less and activity againstgalactose of 5% or less, with respect to the activity against glucosewhich is assumed to be 100%), such excellent characteristics not beingrealized by conventional ones.

However, the coenzyme-linked glucose dehydrogenase disclosed in PatentDocument 1 is isolated or extracted from liquid culture medium in whichwild microorganisms (such as, for example, microorganisms belonging tothe genus Aspergillus) are cultivated, and therefore, the productionyield thereof is limited. In addition to the slight production yield ofthe enzyme, large amounts of sugar are bound to the enzyme, andtherefore the enzyme is in a so-called “sugar-embedded-type enzyme” fromwhich is coated by different kinds of sugar from N-type or O-type sugarchain which binds to general enzymes, as a result of which the activitythereof is difficult to be detected (that is, the enzyme activity islow), the sugar chain cannot be removed enzymatically or chemically, andthereby, the enzyme is scarcely stained by usual protein staining (usingCoomassie Brilliant Blue G-250 or the like) after electrophoresis, and aterminal or internal amino acid sequence of the enzyme, which isnecessary information for obtaining the gene, is difficult to decode byperforming conventional purification. Accordingly, there is no case inwhich cloning of the enzyme gene succeeds to ascertain expression of theenzyme activity.

Although the existence of coenzyme-linked glucose dehydrogenasesoriginating from Aspergillus oryzae was suggested in 1967 (Non-patentDocument 1), only partial enzymatic properties thereof were revealed.Although the dehydrogenase was suggested to provide no influence onmaltose, there are no detailed reports regarding the coenzyme-linkedglucose dehydrogenases originating from Aspergillus oryzae, and noreports regarding coenzyme-linked glucose dehydrogenases originatingfrom other microorganisms which oxidize a hydroxyl group at the1-position of glucose, and also no reports regarding amino acidsequences or genes of the coenzyme-linked glucose dehydrogenases areknown.

Although an idea of measuring glucose using a glucose dehydrogenase EC1.1.99.10 is known (see Patent Document 15), there is no case in whichany coenzyme-linked glucose dehydrogenases are produced to a practicallevel, and therefore, no coenzyme-linked glucose dehydrogenases havebeen developed for practical use in a sensor. The reason for this is theactivity of the enzyme in the fungus body is weak, and even if theenzyme is secreted outside the fungus body, the amount thereof isextremely slight, and the activity thereof is weak because the enzyme iscoated by a large amount of sugar, as a result of which the enzyme isdifficult to detect. Accordingly, it is speculated that a gene of theenzyme can not be cloned.

It is been known that the measurement of glucose levels using a sensorutilizing a glucose oxidase is influenced by sugar chains of the enzyme,and thereby it is difficult for an enzyme originating from molds rich insugar chains to be adapted to the glucose sensor (Non-patent Document2). It is known, for example, that solid cultivation of microorganismsbelonging to the genus Aspergillus increases the sugar content ofyielded enzymes in comparison with liquid cultivation thereof(Non-patent Document 3), and thus it is known that solid cultivationgenerally increases sugar chains in comparison with liquid cultivation.Thus, one of reasons coenzyme-linked glucose dehydrogenases have notbeen developed for practical use until now is assumed to be because ithas been difficult to reduce sugar chain contents of the glucosedehydrogenases originating from molds to utilize it in a glucose sensoreven if cultivating conditions are investigated.

In fact, although the present inventors purified a coenzyme-linkedglucose dehydrogenase originating from Aspergillus terreus, theinventors found that the obtained dehydrogenase was coated with a greatamount of sugars to be in a form which may be called an “arabinogalactanembedded-type enzyme”, as a result of which an enzyme-immobilizedelectrode formed by applying the enzyme on an electrode and then dryingis not sufficiently dried, and the reactivity of a glucose sensor isdeteriorated by the existence of the sugars.

Biogenetic methods in which gene stocks encoding proteins such asenzymes or the like are utilized to produce the proteins on a massivescale are known, and biogenetic methods for preparing glucosedehydrogenases as disclosed in Patent Documents 2 to 14 are known. Thesemainly relate to modification of PQQ glucose dehydrogenases, and providemodified PQQ glucose dehydrogenases, in which disadvantages ofconventional PQQ glucose dehydrogenases, such as low substrateselectivity and low stability, are improved, and modified gene stocksfor biogenetically preparing the modified PQQ glucose dehydrogenases.

[Patent Document 1] WO2004/058958 Pamphlet [Patent Document 2] JapaneseLaid-Open Patent Application No. 2000-312588 [Patent Document 3]Japanese Laid-Open Patent Application No. 2000-350588 [Patent Document4] Japanese Laid-Open Patent Application No. 2000-354495 [PatentDocument 5] Japanese Laid-Open Patent Application No. 2001-197888[Patent Document 6] Japanese Laid-Open Patent Application No.2001-346587 [Patent Document 7] Japanese Laid-Open Patent ApplicationNo. 2001-37483 [Patent Document 8] Japanese Laid-Open Patent ApplicationNo. 2004-173538 [Patent Document 9] Japanese Laid-Open PatentApplication No. 2004-313172 [Patent Document 10] Japanese Laid-OpenPatent Application No. 2004-313180 [Patent Document 11] JapaneseLaid-Open Patent Application No. 2004-344145 [Patent Document 12]Japanese Unexamined Patent Application, First Publication No. H10-243786[Patent Document 13] Published Japanese translation No. 2004-512047 ofPCT International Publication [Patent Document 14] WO2002/072839Pamphlet [Patent Document 15] Japanese Unexamined Patent Application,First Publication No. S59-25700 [Non-patent Document 1] Biochem.Biophys. Acta., 139, 277-293. 1967 [Non-patent Document 2] Appl EnvironMicrobiol., 64(4), 1405-1411, 1998 [Non-patent Document 3] Biosci.Biotechnol. Biochem., 62(10), 1938-1946, 1998

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, in the case of the modified PQQ glucose dehydrogenases preparedusing the modified gene stocks, the degree of activity toward maltose isapproximately 10% or more, which is high, with respect to the degree ofactivity toward glucose which is assumed to be 100%. When the reactivitytoward maltose is lowered, the reactivity (specific activity) to glucoseis also lowered. Accordingly, when the activity is monitored by anelectrochemical measurement method under a condition in which thecontent of substrate is sufficient, functions as a glucose sensor areinsufficiently exhibited, and practical application to POCT apparatusesor the like has not been realized.

Also, there are disadvantages that a coenzyme PQQ required forexpressing activity of the PQQ glucose dehydrogenase is not produced byEscherichia coli bacterium which is broadly used as a recombinant hostin general, and so the recombinant host thereof is limited to hostmicroorganisms that produce PQQ (such as, for example, Pseudomonad).

The present invention has been achieved in view of the above-mentionedproblems of the prior arts, and has as its object to provide: acoenzyme-linked glucose dehydrogenase, in which the problems caused bythe great deal of sugar bonded to the enzyme are solved, and which hasexcellent characteristics such as an excellent reactivity towardglucose, thermal stability, and substrate-recognizing ability, and a lowactivity toward maltose; a method for easily producing thecoenzyme-linked glucose dehydrogenase on a massive scale; apolynucleotide encoding the dehydrogenase; a method for producing thepolynucleotide; a method for measuring glucose levels using thedehydrogenase; a reagent composition for measuring the glucose levels;and a biosensor for measuring the glucose levels.

Means for Solving the Problems

The inventors of the present invention considered that mass productionof a glucose dehydrogenase that does not act on maltose needs to berealized at a practical cost by gene cloning, in addition to a decreaseof the content of the sugar chain massively bonded to the dehydrogenaseto an applicable level for measurement of glucose, so that thecoenzyme-linked glucose dehydrogenase is broadly utilized for industrialapplication. Moreover, the inventors considered that decoding ofterminal or internal amino acid sequences of the dehydrogenase andobtaining of information necessary for obtaining the gene are essentialfor cloning the gene, and therefore, removal of the great deal of sugarembedding the dehydrogenase, different from general N-type or O-typesugar chains, to improve the stainability of the protein as well as torealize HPLC analysis, is required. Accordingly, the inventors haveearnestly investigated to obtain a purified dehydrogenase from which thesugar embedding the dehydrogenase is removed, the sugar making itdifficult to perform protein staining and HPLC analysis. As a result,the inventors have found that solid cultivation enables the content ofthe sugar embedding the objective dehydrogenase to be reduced, andthereby the amino acid sequence thereof is revealed to obtain the genethereof.

In order to solve the above-mentioned problems, the present inventionprovides a polynucleotide encoding a soluble coenzyme-linked glucosedehydrogenase (GLD) (hereinafter, may be referred to as “GLDpolynucleotide”), characterized by catalyzing dehydrogenation of glucosein the presence of an electron acceptor and exhibiting 5% or less,preferably 3% or less, and more preferably 2% or less, of an activitytoward maltose, with respect to an activity toward glucose.

In more detail, the polynucleotide is as follows.

(A) A GLD polynucleotide is characterized in that the GLD has thefollowing properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) having a subunit structure of a homodimer;

3) not utilizing oxygen as an electron acceptor; and

4) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose.

(B) Alternatively, a GLD polynucleotide is characterized in that the GLDhas the following properties 1) to 3) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) not utilizing oxygen as an electron acceptor; and

3) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose.

(C) Alternatively, a GLD polynucleotide is characterized in that the OLDhas the following properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) not utilizing oxygen as an electron acceptor;

3) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose; and

4) having a total sugar content (galactose, glucose, mannose, andarabinose) of 80 μg or less per μg of a protein.

(D) Alternatively, a GLD polynucleotide is characterized in that the GLDhas the following properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) not utilizing oxygen as an electron acceptor;

3) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose; and

4) having a total sugar content (galactose, glucose, mannose, arabinose)of 40 μg or less per unit of an enzyme activity;

(E) Alternatively, a polynucleotide encoding a coenzyme-linked glucosedehydrogenase includes at least one partial nucleotide sequence selectedfrom consensus sequences of a nucleotide sequence encoding thecoenzyme-linked glucose dehydrogenase, set forth in SEQ ID NOs. 5 to 7,the coenzyme-linked glucose dehydrogenase having the followingproperties a to d of:a having a subunit molecular weight of approximately 63 kDa;b utilizing FAD as a coenzyme;c catalyzing a reaction in which a hydroxyl group at the 1-position of aglucose is oxidized and the glucose is converted to a glucono-δ-lactone;andd having an activity toward maltose of 5% or less, with respect to anactivity toward glucose.

In the above, the term “subunit molecular weight” set forth in theproperty a refers to a subunit molecular weight determined by subjectinga coenzyme-linked glucose dehydrogenase originating from prokaryoticcells in which the GLD polynucleotide with or without its signal peptideregion is subjected to polyacrylamide gel electrophoresis (SDS-PAGE),the subunit molecular weight being within the range of 58 kDa to 63 kDa.When the subunit molecular weight is determined using a coenzyme-linkedglucose dehydrogenase originating from eukaryotic cells, the subunitmolecular weight is within the range of 58 kDa to 150 kDa.

(F) Alternatively, a polynucleotide encoding a coenzyme-linked glucosedehydrogenase includes at least one partial amino acid sequence selectedfrom consensus sequences of the coenzyme-linked glucose dehydrogenaseset forth in SEQ ID NOs. 8 to 12, the coenzyme-linked glucosedehydrogenase having the following properties a to d of:a having a subunit molecular weight of approximately 63 kDa;b utilizing a FAD as a coenzyme;c catalyzing a reaction in which a hydroxyl group at the 1-position of aglucose is oxidized and the glucose is converted to a glucono-δ-lactone;andd having an activity toward maltose of 5% or less with respect to anactivity toward glucose.

In the above, the term “subunit molecular weight” set forth in theproperty a refers to a subunit molecular weight determined by subjectinga coenzyme-linked glucose dehydrogenase originating from prokaryoticcells in which the GLD polynucleotide with or without its signal peptideregion is subjected to polyacrylamide gel electrophoresis (SDS-PAGE),the subunit molecular weight being within the range of 58 kDa to 63 kDa.When the subunit molecular weight is determined using a coenzyme-linkedglucose dehydrogenase originating from eukaryotic cells, the subunitmolecular weight is within the range of 58 kDa to 150 kDa.

The GLD encoded by the GLD polynucleotide is an enzyme that hasphysicochemical properties of catalyzing a reaction in which a hydroxylgroup at the 1-position of glucose is oxidized in the presence of anelectron acceptor with a flavin compound (flavin adenine dinucleotide)as a coenzyme. The GLD exhibits an activity toward maltose of 5% orless, preferably 3% or less, and more preferably 2% or less. Theactivity toward maltose is inhibited by 50% or more in the presence of1,10-phenanthroline at a final concentration of 5 mM, preferably 2 mM,and more preferably 1 mM. Although the subunit structure of the GLD is ahomodimer, there is a case in which a monomer thereof exhibits activity.

The total content of sugar (galactose, glucose, mannose, and arabinose)contained in the GLD encoded by the GLD polynucleotide is different fromthat of a wild type GLD, and is 80 μg or less, preferably 10 μg or less,more preferably 2 μg or less, and even more preferably 0.5 μg or less,per μg of protein.

Also, the total content of sugar (galactose, glucose, mannose, andarabinose) contained in the GLD encoded by the GLD polynucleotide isdifferent from that of a wild type GLD, and is 40 μg or less, preferably10 μg or less, more preferably 2 μg or less, and even more preferably0.5 μg or less, per unit of enzyme activity.

The GLD encoded by the GLD polynucleotide has a subunit molecular weightof approximately 63 kDa, utilizes flavin adenine dinucleotide (FAD) as acoenzyme, catalyzes a reaction in which a hydroxyl group at the1-position of glucose is oxidized and the glucose is converted toglucono-δ-lactone, and has an activity toward maltose of 5% or less withrespect to activity toward glucose.

The GLD polynucleotide is specifically a polynucleotide isolated from afilamentous fungi or a basidiomycete, such as, for example, amicroorganism belonging to the genus Aspergillus Penicillium, or thegenus Ganoderma, and is particularly a polynucleotide isolated fromAspergillus terreus (A. terreus).

The specific aspect of the GLD polynucleotide according to the presentinvention is a polynucleotide containing a nucleotide sequence set forthin SEQ ID NO. 1 or a nucleotide sequence in which at least one base isdeleted from, substituted in, or added to the nucleotide sequence setforth in SEQ ID NO. 1, and encoding the GLD having a glucose dehydrationactivity realized when a coenzyme, particularly FAD, is bonded thereto.

Also, the present invention provides a polynucleotide containing anucleotide sequence with a homology of at least 60% to a polynucleotidecomposed of the nucleotide sequence set forth in SEQ ID NO. 1, andencoding the GLD having a glucose dehydration activity realized when acoenzyme, particularly FAD, is bonded thereto.

The term “nucleotide sequence with a homology of at least 60% to apolynucleotide composed of the nucleotide sequence set forth in SEQ IDNO. 1” refers to a nucleotide sequence of which the identity to thefull-length nucleotide sequence set forth in SEQ ID NO. 1 is at least60%, preferably at least 70%, more preferably at least 80%, even morepreferably at least 90%, and particularly preferably at least 95%. Thepercentage of such a nucleotide sequence identity may be calculatedusing a published or commercially available software with an algorithmwhich conducts comparison using a base sequence (SEQ ID NO. 1 in thepresent invention) as a reference sequence. For example, BLAST, FASTA,or GENETYX (manufactured by Software Development Co., Ltd.) may be used,and these may be run with default parameters.

Also, the present invention provides a polynucleotide containing anucleotide sequence which hybridizes under stringent conditions to apolynucleotide composed of a nucleotide sequence complementary to apolynucleotide composed of the nucleotide sequence set forth in SEQ IDNO. 1, and encoding the GLD having a glucose dehydration activityexhibited by binding a coenzyme, particularly FAD.

The first amino acid Met to the 19th amino acid Leu of the GLD encodedby the nucleotide sequence form a signal peptide thereof. Apolynucleotide encoding this region may be suitably substituted ordeleted depending on organisms or host vector systems.

Also, the present invention provides a method for producing thepolynucleotide encoding the GLD, in which a microorganism having a GLDproductivity is cultivated in a solid state, and the polynucleotide iscloned based on an information of the dehydrogenase produced. It ispreferable that the microorganism used be at least one strain belongingto the genus Aspergillus, particularly Aspergillus terreus (A. terreus).

Moreover, the present invention provides the GLD encoded by any one ofthe above-mentioned polynucleotide nucleotide sequences. A more specificaspect of the GLD according to the present invention is a soluble GLDwhich catalyzes dehydrogenation of glucose in the presence of anelectron acceptor, and has an activity toward maltose of 5% or less withrespect to an activity toward glucose.

Also, the GLD has the following properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide as a coenzyme;

2) having a subunit structure of a homodimer;

3) not utilizing oxygen as an electron acceptor; and

4) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose.

Alternatively, the GLD has the following properties 1) to 3) of:

1) utilizing flavin adenine dinucleotide as a coenzyme;

2) not utilizing oxygen as an electron acceptor; and

3) having an activity toward maltose of 5% or less, preferably 3% orless, and more preferably 2% or less, with respect to an activity towardglucose.

Alternatively, the GLD has the following properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) not utilizing oxygen as an electron acceptor;

3) having an activity toward maltose of 5% or less with respect to anactivity toward glucose; and

4) having a total content of galactose, glucose, mannose, and arabinose,contained therein, of 80 μg or less per μg of a protein.

Alternatively, the GLD has the following properties 1) to 4) of:

1) utilizing flavin adenine dinucleotide (FAD) as a coenzyme;

2) not utilizing oxygen as an electron acceptor;

3) having an activity toward maltose of 5% or less with respect to anactivity toward glucose; and

4) having a total content of galactose, glucose, mannose, and arabinose,contained therein, of 40 μg or less per unit of an enzyme activity.

Alternatively, the GLD has the following properties of:

a. having a subunit molecular weight of approximately 63 kDa;

b utilizing a FAD as a coenzyme;

c catalyzing a reaction in which a hydroxyl group at the 1-position of aglucose is oxidized and the glucose is converted to a glucono-δ-lactone;and

d having an activity toward maltose of 5% or less with respect to anactivity toward glucose.

The GLD is isolated from filamentous fungi, preferably at least onestrain belonging to the genus Aspergillus, and more preferablyAspergillus terreus.

A more specific aspect of the GLD according to the present invention isa GLD containing an amino acid sequence set forth in SEQ ID NO. 2, andwhich dehydrates glucose by binding to a coenzyme, particularly FAD.Also, a GLD containing an amino acid sequence in which at least oneamino acid is deleted from, substituted in, or added to, the amino acidsequence set forth in SEQ ID NO. 2, and has a glucose dehydrationactivity exhibited by binding FAD is provided.

Also, the present invention provides a GLD containing an amino acidsequence with a sequence homology of at least 60% to the amino acidsequence set forth in SEQ ID NO. 2, and has a glucose dehydrationactivity exhibited by binding a coenzyme, particularly FAD. Althoughcoenzyme-linked glucose dehydrogenases originating from drosophila havebeen conventionally known (Proc. Natl. Acad. Sci. 1983 October; 80:6286-6288, “Biphasic expression and function of glucose dehydrogenase inDrosophila melanogaster.”), the homology thereof to the amino acidsequence set forth in SEQ ID NO. 2 is 45 to 47%. Moreover, it isdifficult for such a dehydrogenase originating from insects to beexpressed in cells other than insect cells, and therefore theproductivity thereof is very low. Accordingly, it is difficult for it tobe industrially utilized. The dehydrogenase according to the presentinvention, containing an amino acid sequence with a sequence homology ofat least 60% to the amino acid sequence set forth in SEQ ID NO. 2, canbe expressed in an Escherichia coli bacterium or the like, andtherefore, is easily utilized as an enzyme for industrial application.

Also, the present invention provides a polynucleotide encoding the GLDcontaining the amino acid sequence set forth in SEQ ID NO. 2.

The term “amino acid sequence with a sequence homology of at least 60%to the amino acid sequence set forth in SEQ ID NO. 2” refers to an aminoacid sequence of which the identity to the full-length amino acidsequence set forth in SEQ ID NO. 2 is at least 60%, preferably at least70%, more preferably at least 80%, even more preferably at least 90%,and particularly preferably at least 95%. The percentage of such anamino acid sequence identity may be calculated using a published orcommercially available software with an algorithm which conductscomparison using a base sequence (SEQ ID NO. 2 in the present invention)as a reference sequence. For example, BLAST, FASTA, or GENETYX(manufactured by Software Development Co., Ltd.) may be used, and thesemay be run with default parameters.

Also, the present invention provides a method for producing the GLD,characterized in that a microorganism that produces any one of theabove-mentioned GLDs is cultivated in a solid culture medium containinga wheat bran or an oatmeal to make the GLD produced in the cultivatedproduct, and then the GLD is collected, and provides the OLD produced bythe method.

Moreover, the present invention provides a recombinant vector carryingany one of the above-mentioned polynucleotides according to the presentinvention, a transformed cell prepared using the recombinant vector, amethod for producing the GLD characterized in that the transformed cellis cultivated followed by collecting the GLD having a glucosedehydration activity from the cultivated product, and the OLD producedby the method.

To the OLD produced by such a method, no sugar chain is bonded. Even ifthe sugar chain is bonded to the OLD, it is generally an N-type orO-type sugar chain, and the bonding amount thereof is smaller than thatof a wild type GLD. Also, the sugar chain is easily removed, and the GLDexhibits a high activity.

Also, the present invention provides a OLD containing an amino acidsequence set forth in amino acid 20 to amino acid 592 of SEQ ID NO. 2 oran amino acid sequence with a homology of at least 60% to the amino acidsequence, having a function equivalent to that of the above-mentionedGLD, and being produced by a peptide synthesis method or a generecombinant method.

Moreover, the present invention provides a method for measuring glucosecharacterized by utilizing the above-mentioned GLD according to thepresent invention, a reagent composition for measuring glucosecharacterized by containing the above-mentioned GLD, and a biosensor formeasuring glucose characterized by utilizing the above-mentioned GLD. Ina preferable aspect of these, an electron acceptor, particularlyferricyanide, is utilized at a final concentration of within the rangeof 2 mM to 500 mM.

The term “polynucleotide” refers to a molecule in which at least 100phosphoric esters of nucleosides in which purine or pyrimidine isconnected to sugar with a 13-N-glycosidic linkage, such as, ATP(adenosine triphosphate), GTP (guanosine triphosphate), CTP (cytidinetriphosphate), UTP (uridine triphosphate); or dATP (deoxyadenosinetriphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidinetriphosphate), and dTTP (deoxythymidine triphosphate) are bonded. Inmore detail, the term “polynucleotide” includes genomic DNAs encodingthe GLD, mRNAs transcripted from the genomic DNAs, cDNAs synthesizedfrom the mRNAs, and polynucleotides obtained by PCR amplification usingthe mRNAs as templates. The term “oligonucleotide” refers to a moleculein which 2 to 99 nucleotides are linked together. The term “polypeptide”refers to a molecule composed of at least 30 amino acid residues bindingtogether with an amide linkage (peptide linkage) or with a linkage ofunnatural residues, and further includes ones to which sugar chains areadded, and ones in which chemical modification is artificiallyconducted.

Other terms in this specification or concepts of the present inventionwill be circumstantially explained in the section of BEST MODE FORCARRYING OUT THE INVENTION and EXAMPLES. Also, various techniques usedfor carrying out this invention are easily and reliably performed bythose skilled in the art based on known documents or the like exceptingtechniques of which citations are indicated. For example, geneticengineering and molecular biological techniques can be performed inaccordance with methods disclosed in Sambrook and Maniatis, in MolecularCloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork, 1989; Ausubel, F. M. et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1995, or the like, methodsdisclosed in documents cited therein, substantially equivalent methods,modified methods, or the like. The terms used in this specification aremainly based on IUPAC-IUB Commission on Biochemical Nomenclature orconventionally used in the art.

Effects of the Invention

The enzyme according to the present invention is a coenzyme-linkedglucose dehydrogenase (GLD) of which the sugar content is reduced to thelevel that enables the GLD to be applied to a glucose sensor, the sugarcontent contained in a natural enzyme being a great amount, the GLDhaving excellent properties in terms of substrate-recognizing abilityagainst glucose and low activity toward maltose. Also, the method forproducing the dehydrogenase according to the present invention canproduce the dehydrogenase uniformly on a massive scale.

The sugar content of the GLD artificially produced in such a way, thesugar content being an issue of coenzyme-linked glucose dehydrogenasesthat dehydrate glucose by binding FAD, can be controlled in accordancewith objects. Accordingly, it is possible to modify the activity towardsugar (such as glucose) contained in samples to measure the blood sugarby preparing a dehydrogenase of which the sugar content is decreased.

The GLD according to the present invention does not substantially affectmaltose at the time of measuring of the blood sugar, and therefore, theGLD can be applied to a high-precision SMBG apparatus, and greatlycontributes to self-care-medication of diabetes patients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of sugar chain staining performed after cuttingsugar chains of dehydrogenases and then subjecting them to SDS-PAGE.

FIG. 2 shows results of CBB staining performed after cutting sugarchains of dehydrogenases and then subjecting them to SDS-PAGE.

FIG. 3 shows results of activity staining performed after cutting sugarchains of dehydrogenases and then subjecting them to native-PAGE.

FIG. 4 shows results of measurement of sensor characteristics(bimolecular reaction rate constants) of dehydrogenases using an osmiumcomplex as an electron acceptor.

FIG. 5 shows results of measurement of sensor characteristics(bimolecular reaction rate constants) of dehydrogenases using a quinonecompound as an electron acceptor.

FIG. 6 shows results of quantitative analysis performed on D-glucoseusing enzyme-immobilized electrodes.

BEST MODE FOR CARRYING OUT THE INVENTION

The GLD polynucleotide (gene) according to the present invention is apolynucleotide encoding a soluble GLD characterized by catalyzingoxidation of glucose in the presence of an electron acceptor, and havingan activity toward maltose of 5% or less, preferably 3% or less, andmore preferably 2% or less, with respect to an activity toward glucose.In more detail, the polynucleotide is the GLD polynucleotidecharacterized by having any one of the above-mentioned properties (A) to(F).

The most specific embodiment of the GLD polynucleotide according to thepresent invention is a polynucleotide containing a nucleotide sequenceset forth in SEQ ID NO. 1. The polynucleotide is a GLD polynucleotideoriginating from a filamentous fungi, such as, for example, the genusAspergillus, particularly Aspergillus terreus (FERM BP-08578), andencodes the GLD containing an amino acid sequence set forth in SEQ IDNO. 2.

The GLD polynucleotide may be obtained by preparing a cDNA library fromAspergillus terreus (FERM BP-08578), for example, and then determiningthe N-terminal or internal amino acid sequence of the GLD by Edman'smethod, followed by screening the cDNA library using pluraloligonucleotide probes prepared based on the amino acid sequence.

A GLD collected from cultivated products obtained by cultivating atleast one microorganism that can yield the GLD according to the presentinvention, such as, for example, at least one strain selected from thegroup consisting of Aspergillus terreus, Aspergillus japonicus (A.japonicus), and Aspergillus oryzae (A. oryzae), which belong to thegenus Aspergillus, in a solid culture medium containing a wheat bran,oatmeal, or the like, has a low amount of sugar chains binding to theGLD, and therefore, can be easily purified by removing the sugar chains.Accordingly, it is preferable that the GLD obtained by solid cultivationbe used to determine the N-terminal or internal sequence of the GLD.

When a wheat bran is used, a solution containing 40 to 70% by mass ofthe wheat bran is sterilized, 0.5 to 2% by mass of a seed culture liquidis added thereto, and cultivated at room temperature, followed byextracting a GLD crude enzyme from the obtained cultivated fungus body,for example. When an oatmeal is used, a solution containing 40 to 70% bymass of the oatmeal is sterilized, 0.5 to 2% by mass of a seed cultureliquid is added thereto, and cultivated at room temperature, followed byextracting a GLD crude enzyme from the obtained cultivated fungus body,for example.

Although the probe may be labeled by a radioisotope (RI) method ornon-radioisotope method, the probe is preferably labeled by thenon-radioisotope method. As the non-radioisotope method, a fluorescentlabeling method, biotin labeling method, chemiluminescence method, orthe like may be adopted, and the fluorescent labeling method ispreferably used. As the fluorescent substance, a substance which canbind to a base portion of an oligonucleotide may be suitably selected,and examples thereof include cyanine pigments (such as, for example, Cy3and Cy5 of Cy Dye™ series), rhodamine 6G reagents,N-acetoxy-N2-acetylaminofluorene (AAF), AAIFs (iodide derivatives ofAAF), and the like.

Alternatively, the objective OLD gene may be obtained by a PCR method inwhich the cDNA library derived from Aspergillus terreus (FERM BP-08578)is used as a template and a set of the oligonucleotide primer (probe)prepared in the above is used, or by a RT-PCR method in which a wholeRNA or mRNA extracted from Aspergillus terreus (FERM BP-08578) is usedas a template. The upstream region of the cDNA may be amplified by a5′RACE-PCR method using a primer with an oligonucleotide sequence setforth in the 5′ side of SEQ ID NO. 1, and the downstream region of thecDNA may be amplified by a 3′RACE-PCR method using a primer with anoligonucleotide sequence set forth in the 3′ side of SEQ ID NO. 1. Theprimer is preferably designed to have a length (base number) of 15 to 40bases, more preferably 15 to 30 bases, in order to satisfactorilyrealize specific annealing thereof to the template DNA. In the casewhere the primers are used for conducting a LA (long and accurate) PCR,the primers with a length of at least 30 bases are effectively used. Aset or a pair (two) of a sense chain (5′ terminal side) and an antisensechain (3′ terminal side) is constructed so that both primers do notcontain complementary sequences thereto for preventing both primers fromannealing together. Moreover, GC content of the primers is set to beapproximately 50% so as to prevent uneven distribution of GC-richportions or AT-rich portions in the primers to realize stable binding.Since the annealing temperature depends on Tm (melting temperature), theprimers whose Tm values are approximate to each other within the rangeof 55 to 65° C. are selected so as to obtain a PCR product with a highspecificity. Also, it is to be noted that the final concentration of theprimers used in PCR be within the range of approximately 0.1 toapproximately 1 μM. A commercially available software for designingprimers, such as, for example, Oligo™ (manufactured by NationalBioscience Inc. (US)), or GENETYX (manufactured by Software DevelopmentCo., Ltd.) may be used.

The above-mentioned set of the oligonucleotide probe or oligonucleotideprimer may be prepared by cutting the above-mentioned GLD cDNA using asuitable restriction enzyme, or by synthesizing in vitro by a well-knownchemosynthesis technique as disclosed in documents (such as, forexample, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997)Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med.19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979)Meth. Enzymol. 68: 90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage(1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066).

The polynucleotide according to the present invention is composed of anucleotide sequence with a homology of at least 60% to that set forth inSEQ ID NO. 1, and may encode the GLD that exhibits a glucose dehydrationactivity by binding to the coenzyme, particularly FAD.

The polynucleotide according to the present invention may have at leastone base deleted from, substituted in, or added to the nucleotidesequence set forth in SEQ ID NO. 1, and may encode the GLD that realizesa glucose dehydration activity by binding to the coenzyme, particularlyFAD.

The polynucleotide according to the present invention may have acapability of hybridization to DNA complementary to the nucleotidesequence set forth in SEQ ID NO. 1, or DNA with a nucleotide sequencecomplementary to that set forth in SEQ ID NO. 1, under stringentconditions, and also may encode the GLD that exhibits a glucosedehydration activity by binding to the coenzyme, particularly FAD.

The polynucleotide according to the present invention may have at leastone partial nucleotide sequence selected from consensus sequences in thenucleotide sequence encoding the coenzyme-linked glucose dehydrogenase,set forth in SEQ ID NOs. 5 to 7. The polynucleotide according to thepresent invention may encode an enzyme having at least one partial aminoacid sequence selected from consensus sequences in the coenzyme-linkedglucose dehydrogenase, set forth in SEQ ID NOs. 8 to 12. The enzymehaving such a consensus sequence (amino acid sequence) often exhibits anactivity of the GLD according to the present invention, and therefore,such a portion is assumed to form an active center of the enzymeaccording to the present invention.

Regarding three characters “Xaa” in SEQ ID NO. 8, the first character“Xaa” represents amino acid “Ala” or “Gly”, the second character “Xaa”represents amino acid “Ala” or “Val”, and the third character “Xaa”represents “Ile” or “Val”. Regarding four characters “Xaa” in SEQ ID NO.9, the first character “Xaa” represents amino acid “Ala” or “Val”, thesecond character “Xaa” represents amino acid “Ile” or “Leu”, the thirdcharacter “Xaa” represents amino acid “Ala” or “Ser”, and the fourthcharacter “Xaa” represents amino acid “Glu” or “Gin”. Regarding threecharacters “Xaa” in SEQ ID NO. 10, the first character “Xaa” representsamino acid “Ala” or “Leu”, the second character “Xaa” represents aminoacid “Ile” or “Leu”, and the third character “Xaa” represents amino acid“Ile” or “Val”. Regarding three characters “Xaa” in SEQ ID NO. 12, thefirst character “Xaa” represents amino acid “Ala” or “Ser”, the secondcharacter “Xaa” represents amino acid “Asn” or “Ser”, and the thirdcharacter “Xaa” represents amino acid “Ile” or “Val”.

The polynucleotides encoding such an enzyme similar to OLD (OLD-likeenzyme) may be prepared by modifying the above-mentioned GLD cDNAderived from Aspergillus terreus in accordance with a knownmutation-introduction method, mutation-introduction PCR method, or thelike. Alternatively, the polynucleotide may be obtained by a probehybridization method using an oligonucleotide prepared based on aninformation of the nucleotide sequence set forth in SEQ ID NO. 1 fromgenomic DNAs of microorganisms other than Aspergillus terreus or cDNAlibraries thereof. The polynucleotides encoding the GLD-like enzyme canbe obtained by varying stringent conditions for hybridization. Thestringent conditions are defined by salt concentration at ahybridization step and washing step, concentration of an organic solvent(formaldehyde or the like), temperature conditions, or the like, andvarious conditions known by a person skilled in the art, such as, forexample, those disclosed in U.S. Pat. No. 6,100,037 or the like, may beadopted.

In more specific hybridization conditions, a filter is incubated at 42°C. with a mixture composed of 50% formamide, 5×SSC (150 mM sodiumchloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mMethylenediamine tetraacetic acid, pH 7.2), 5×Denhardt's solution, 0.1%SDS, 10% dextran sulfate, and 100 μg/mL of a modified salmon sperm DNA,and then washed at 42° C. with 0.2×SSC, for example.

The species or the genus of a microorganism used to obtain thepolynucleotide encoding the GLD-like enzyme is not limited, and themicroorganism may be a wild strain or a mutant strain. Examples thereofinclude a microorganism disclosed in Patent Document 1.

The recombinant vector according to the present invention is a cloningvector or an expression vector, and is suitably used in accordance withthe kind of an insert polynucleotide and the application purposethereof. For example, when the GLD or the GLD-like enzyme is producedusing the cDNA or ORF region thereof as an insert, expression vectorsfor in vitro transcription, or expression vectors suitable toprokaryotic cells, such as, for example, Escherichia coli bacterium, orBacillus subtilis, yeasts, filamentous fungi, such as, for example,molds, eukaryotic cells, such as, for example, insect cells, ormammalian cells, may be used.

When the GLD or the GLD-like enzyme is produced on a massive scale, thetransformed cell according to the present invention may be preparedusing a prokaryotic cell such as Escherichia coli bacterium, Bacillussubtilis, or the like, yeast, mold, eukaryotic cell, such as insectcells, mammalian cells, or the like, for example. The transformed cellmay be prepared by introducing the recombinant vector into cells by aknown method such as an electroporation method, calcium phosphatemethod, liposome method, DEAE dextran method, or the like. Specificexamples of the recombinant vector include a recombinant vector pCGLDshown in the following example, and specific examples of the transformedcell include an Escherichia coli JM109/pCGLD (FERM ABP-10243) preparedby transformation using the vector.

The GLD according to the present invention is a polypeptide having anamino acid sequence encoded by the above-mentioned GLD polynucleotidesequence. In more detail, it is preferable that the GLD be a solublecoenzyme-linked glucose dehydrogenase which catalyzes dehydrogenation ofglucose in the presence of an electron acceptor, has an activity towardmaltose of 5% or less with respect to an activity toward glucose, andfurther has any one of the above-mentioned properties (A) to (F).

In a more specific aspect of the GLD according to the present invention,the total content of sugars (galactose, glucose, mannose, and arabinose)contained therein is 80 μg or less per μg of a protein, or 40 μg or lessper unit of an enzyme activity. The sugars form polysaccharides bypolycondensation and envelope the enzyme. Accordingly, when the totalcontent of the sugars contained therein is 80 μg or less per μg of aprotein, or 40 μg or less per unit of an enzyme activity, the enzymewith a high activity can be obtained, and thus such a total content ispreferable.

The more specific aspect of the GLD according to the present inventionis composed of an amino acid sequence set forth in SEQ ID NO. 2. The GLDaccording to the present invention may also be a GLD-like enzymecomposed of an amino acid sequence with a homology of at least 60% tothat set forth in SEQ ID NO. 2, the GLD-like enzyme exhibiting a glucosedehydration activity by binding to a coenzyme, particularly FAD. The GLDaccording to the present invention may be a GLD-like enzyme composed ofan amino acid sequence with at least one amino acid residue deletedfrom, substituted in, or added to the amino acid sequence set forth inSEQ ID NO. 2, the GLD-like enzyme exhibiting a glucose dehydrationactivity by binding to a coenzyme, particularly FAD. The GLD accordingto the present invention may be a polypeptide that has either an aminoacid sequence set forth in amino acid 20 to amino acid 592 of SEQ ID NO.2 or an amino acid sequence with a homology of at least 60% to the aminoacid sequence, exhibits a function equivalent to that of theabove-mentioned polypeptide, and is synthesized by a peptide synthesismethod or gene recombinant method.

Such a GLD may be prepared, for example, based on the amino acidsequence set forth in SEQ ID NO. 2 or a similar sequence thereto by aknown peptide synthesis method (Merrifield, R. B. J. Solid phase peptidesynthesis I. The synthesis of tetrapeptide. J. Amer. Chem. Soc. 85,2149-2154, 1963; Fmoc Solid Phase Peptide Synthesis. A PracticalApproach. Chan, W. C. and White, P. D., Oxford University Press, 2000).The peptide may be formed by a residue linkage other than natural amidelinkages. The residue linkage other than natural amide linkages may beformed by a chemical binding or coupling using glutaraldehyde,N-hydroxysuccinimide ester, bifunctional maleimide,N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),or the like. Examples of a linkage group which can be substituted for apeptide binding include ketomethylene (for example, —C(═O)—CH₂— insteadof —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether(CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide,thioamide, and ester (see, for example, Spatola (1983) in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide Backbone Modifications” Marcell Dekker, NY).

The GLD may be obtained by a recombinant DNA technique using theabove-mentioned GLD polynucleotide (cDNA or the coding region thereof).For example, RNA is prepared by in vitro transcription using a vectorcontaining the above-mentioned polynucleotide, followed by subjectingthe RNA as a template to in vitro translation to produce the GLD invitro. When the polynucleotide is inserted into a suitable expressionvector by a known-method, the GLD encoded by the polynucleotide can beyielded on a massive scale using prokaryotic cells such as Escherichiacoli bacterium, Bacillus subtilis, or the like, yeasts, molds,eukaryotic cells such as insect cells, mammalian cells, or the like. Ahost to be used is suitably selected in accordance with necessity or notof sugar chains, and necessity of other peptide modification.

In order to produce the GLD in vitro, a recombinant vector is preparedby inserting the above-mentioned polynucleotide into a vector containinga promoter to which RNA polymerase can be bonded, followed by adding thevector to an in vitro translation system such as rabbit bloodreticulocyte lysate or wheat germ extract, which contain RNA polymeraseresponding to the promoter. Examples of the promoter to which RNApolymerase can be bonded include T3, T7, SP6, and the like. Examples ofthe vector containing such a promoter include pKA1, pCDM8, pT3/T718,pT7/319, pBluescript II, and the like.

In the case where the GLD is produced by expressing DNA thereof in amicroorganism such as Escherichia coli bacterium or the like, the GLDcan be produced on a massive scale in the microorganism by preparing arecombinant expression vector in which the above-mentionedpolynucleotide is inserted into an expression vector replicable in themicroorganism, the expression vector having an origin, promoter,ribosome binding site, DNA cloning site, terminator sequence, and thelike, followed by transforming host cells with the recombinantexpression vector, and then cultivating the transformed cells. In thiscase, a OLD fraction containing an optional region can be obtained byadding an initiation codon and a stop codon in front of and behind acoding region of the optional region, followed by expressing theoptional region. Alternatively, the GLD may be expressed as a proteinfused with another protein. The fused protein may be cut with a suitableprotease to obtain an objective GLD. Examples of the expression vectorused in an Escherichia coli bacterium include pUC vectors, pBluescriptII, pET expression vectors, pGEX expression vectors, pCold expressionvectors, and the like.

The GLD can be produced in a eukaryotic cell by preparing a recombinantvector in which the above-mentioned polynucleotide is inserted into anexpression vector replicable in eukaryotic cells, the expression vectorhaving a promoter, splicing region, polyA-addition site, and the like,followed by transforming the eukaryotic cells with the recombinantvector. The recombinant vector may be held in the cells in such a stateas that of a plasmid or may be held by incorporating it in a chromosome.Examples of the expression vector include pKA1, pCDM8, pSVK3, pSVL,pBK-CMV, pBK-RSV, EBV vector, pRS, pYE82, and the like. WhenpINDN/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or the like is used asthe expression vector, FAD-GLD polypeptide can be expressed as a fusedprotein to which a tag, such as His-tag, FLAG-tag, OFP or the like, isadded. Although mammalian culture cells, such as monkey kidney cellsCOS-7, Chinese hamster ovary cells CHO, or the like, budding yeasts,fission yeasts, molds, silkworm cells, xenopus oocytes, are generallyused as the eukaryotic cells, any eukaryotic cells may be used providedthat they can yield the GLD. In order to introduce the expression vectorinto the eukaryotic cell, a known method such as an electroporationmethod, calcium phosphate method, liposome method, DEAE dextran method,or the like, may be adopted.

After the GLD is yielded in prokaryotic cells or eukaryotic cells, theobjective protein is purified following isolation from cultivatedproducts (such as, for example, fungus body, or cultivated liquid orculture medium composition, containing the enzyme secreted outward ofthe fungus body) by combining known separation procedures. Examples ofsuch procedures include a treatment using a denaturant such as urea or asurfactant, thermal treatment, pH treatment, sonication, enzymaticdigestion, salting-out or solvent precipitation method, dialysis,centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectricfocusing electrophoresis, ion-exchange chromatography, hydrophobicchromatography, reversed-phase chromatography, affinity chromatography(including a method in which a tag sequence is utilized and a method inwhich a polyclonal or monoclonal antibody that is specific to UKC1 isutilized).

The GLD according to the present invention, which is prepared by theabove-mentioned method, has the following characteristics.

(1) Action: The GLD is an enzyme classified in class EC 1.1.99.10 byInternational Union of Biochemistry (IUB) and catalyzes reaction inwhich a hydroxyl group at the 1-position of glucose is oxidized in thepresence of an electron acceptor to produce glucono-δ-lactone(glucose+electron acceptor→glucono-δ-lactone+reduced-form electronacceptor).

Examples of the electron acceptor for use include phenazinemethosulfate,1-methoxy-5-methylphenazium methylsulfate, 2,6-dichlorophenolindophenol,ferricyanide compounds, osmium compounds, quinone compounds, and thelike.

(2) Substrate selectivity: The OLD strongly acts on D-glucose, butweakly acts on D-mannose, 1,5-anhydro-D-glucitol, D-cellobiose,D-trehalose, maltose, D-galactose, D-glucose-6-phosphate, andD-fructose. The GLD exhibits almost no action on L-arabinose, lactose,D-sorbitol, gluconic acid, sucrose, D-mannitol, L-sorbose, D-ribose,L-rhamnose, D-glucose-1-phosphate, D-raffinose, ethanol, and glycerol.(3) Inhibitor: At least 60% of the activity is inhibited by1,10-phenanthroline.(4) Coenzyme: Flavin adenine dinucleotide(5) Optimum pH: 7.0 to 9.0(6) Stable pH: 4.5 to 8.5(7) Optimum temperature: approximately 55° C.(8) Temperature stability: The GLD is stable at approximately 50° C. orlower.

Since sugar chains attach to the enzyme, the above-mentioned molecularweight thereof varies in accordance with cultivating conditions orpurification conditions. In the case of the recombinant, the kind ofsugar chain or amino acid attaching thereto varies in accordance withthe kind of the host or vector used, and therefore, the molecular weightthereof also varies.

It is ascertained that the isoelectric focusing also varies in a similarmanner to the above.

As described above, the GLD according to the present invention is anenzyme which catalyzes dehydrogenation of glucose in the presence of anelectron acceptor, and therefore, the use thereof is not particularlylimited, provided that it utilizes the change caused by thedehydrogenation. For example, the OLD may be used in medical fields orclinical fields for measuring glucose in samples containingbiomaterials, or for composing a reagent for measuring glucose or areagent for eliminating glucose. Alternatively, the GLD may be used forproducing a substance using a coenzyme-linked glucose dehydrogenase.

The biosensor according to the present invention contains the GLDaccording to the present invention as an enzyme in a reaction layer, andis a glucose sensor for measuring glucose concentration in sampleliquids. For example, the biosensor is prepared by forming an electrodesystem having a working pole, counter pole thereof, and reference pole,on an insulating base plate by a screen printing method or the like,followed by forming an enzyme reaction layer containing a hydrophilicpolymer, oxidoreductase, and electron acceptor, onto the electrodesystem. When a sample liquid containing a substrate is dropped onto theenzyme reaction layer of the biosensor, the enzyme reaction layer isdissolved, and then the substrate is reacted with the enzyme, as aresult of which the electron acceptor is reduced. After the enzymereaction is ended, the reduced electron acceptor is electrochemicallyoxidized to measure the oxidation current value. The concentration ofthe substrate in the sample liquid is determined by the oxidationcurrent value. In addition to the above, a biosensor that detects changeof developed color or pH may be constructed.

As the electron acceptor of the biosensor, chemical substances that havean excellent electron donating and accepting ability may be used. Theterm “chemical substances that have an excellent electron donating andaccepting ability” refer to chemical substances generally called an“electron carrier”, “mediator” or “redox mediator”, and examples thereofinclude electron carriers and redox mediators disclosed in PublishedJapanese translation No. 2002-526759 of PCT International Publication.Specific examples thereof include osmium compounds, quinone compounds,ferricyanide compounds, and the like.

As the electron acceptor of the biosensor, cheap potassium ferricyanide(potassium hexacyanoferrate (III)) is often used at the finalconcentration of 1 mM or less. However, D-glucose can be furthersensitively measured by using potassium ferricyanide in a highconcentration of 2 to 500 mM, and more preferably 30 to 100 mM. It ispreferable that potassium ferricyanide be used at the finalconcentration of 2 to 500 mM in the measurement reaction system of themeasurement method, reagent for measurement, compound for measurement,or biosensor, according to the present invention.

In measurement of the activity of the enzyme according to the presentinvention, it is preferable that the enzyme be suitably diluted to thefinal concentration of 0.1 to 1.0 unit/ml, for use. One unit of theenzyme activity is equivalent to the enzyme activity that oxidizes 1μmol of glucose per minute. The enzyme activity of the coenzyme-linkedglucose dehydrogenase according to the present invention may bedetermined in accordance with the following method.

(i) Enzyme Activity Measurement Method-1

1.0 ml of 0.1 M potassium phosphate buffer (pH 7.0), 1.0 ml of 1.0 MD-glucose, 0.1 ml of 3 mM 2,6-dichlorophenolindophenol (hereinafter,referred to as DCIP), 0.2 ml of 3 mM 1-methoxy-5-methylphenaziummethylsulfate, and 0.65 ml of water are placed in a 3 ml quartz cell(with an optical path length of 1 cm), and then the cell is placed in aspectrophotometer equipped with a thermostat cell holder. After the cellis incubated at 37° C. for five minutes, 0.05 ml of an enzyme liquid isadded thereto, followed by measuring absorbance change (ΔABS/min) ofDCIP at 600 nm. The mol-absorption coefficient of DCIP at pH 7.0 isdefined to be 16.3×10³ cm⁻¹M⁻¹. Since one unit of the enzyme activity issubstantially equivalent to the enzyme activity that reduces 1 μmol ofDCIP per minute, the enzyme activity is determined by absorbance changein accordance with the following formula.

${{Enzyme}\mspace{14mu}{activity}\mspace{14mu}\left( {{unit}\text{/}{ml}} \right)} = {\frac{{- \Delta}\;{ABS}}{16.3} \times \frac{3.0}{0.05} \times {dilution}\mspace{14mu}{rate}\mspace{14mu}{of}\mspace{14mu}{enzyme}}$(ii) Enzyme Activity Measurement Method-2

After 3.4 μl of 1.0 M potassium phosphate buffer (pH 7.0), 0.1 ml of 1.0M D-glucose, and 86.6 μl of 20 mM DCIP are incubated at 37° C. for fiveminutes, 0.01 ml of an enzyme liquid is added thereto and then stirredto react the mixture for five minutes, followed by incubating themixture for three minutes at 100° C. to stop the reaction. Moreover,0.19 ml of 100 mM glycine/sodium buffer (pH 13.0) and 0.01 ml of 2.0 Npotassium hydroxide are further added and incubated at 37° C. for tenminutes to convert D-gluconic acid in the mixture toD-glucono-δ-lactone, followed by adding 0.39 ml of 100 mMTris-hydrochloride buffer (pH 7.5) and 0.01 ml of 1.0N hydrochloric acidthereto to obtain a neutral pH. The amount of D-gluconic acid in themixture is quantitatively analyzed using a D-gluconicacid/D-glucono-δ-lactone quantitative analysis kit (manufactured byRoche Diagnostics K.K.). Since the enzyme activity that produces oneμmol of D-glucono-δ-lactone per minute is substantially equivalent toone unit of the enzyme activity, the enzyme activity is determined basedon the yield amount of D-glucono-δ-lactone.

In measurement of the protein concentration of the enzyme, the enzyme ispreferably used by suitably diluting it to the final concentration of0.2 to 0.9 mg/ml. The protein concentration may be determined by using akit for measuring the protein concentration, purchased from Bio-RadLaboratories, Inc., under the trade name of Bio-Rad Protein Assay, inaccordance with an instruction manual, and calculating using a standardcurve drawn by using bovine serum albumin (BSA, manufactured by WakoPure Chemical Industries, Ltd., biochemical reagent) as a standardsubstance.

In the following, the present invention will be further circumstantiallyexplained by showing some examples. However, the present invention isnot limited to the following examples.

Example 1

1-1 (Seed Cultivation)

A pH of a liquid culture medium composed of 1% (W/V) glucose(manufactured by Wako Pure Chemical Industries, Ltd.), 2% (W/V) defattedsoybean (manufactured by Nihon Syokuhan Co., Ltd.), 0.5% (W/V) cornsteep liquor (manufactured by San-ei Sucrochemical Co., Ltd.), 0.1%(W/V) magnesium sulfate heptahydrate (manufactured by Nacalai Tesque,Inc.) and water was adjusted to 6.0. 100 mL of the liquid culture mediumwas placed in a Sakaguchi flask of 500 ml capacity, and plugged withcotton, followed by performing autoclave treatment at 121° C. for 20minutes. After the culture medium was cooled, a strain of Aspergillusterreus (FERM BP-08578) was inoculated thereto, followed by cultivatingwhile shaking at 28° C. for 48 hours to obtain a seed culture liquid.

1-2 (Obtaining Crude Enzyme Liquid by Liquid Cultivation)

A pH of 4 L of a liquid culture medium composed of 1% (W/V) glucose(manufactured by Wako Pure Chemical Industries, Ltd.), 2% (W/V) defattedsoybean (manufactured by Nihon Syokuhan Co., Ltd.), 0.5% (W/V) cornsteep liquor (manufactured by San-ei Sucrochemical Co., Ltd.), 0.1%(W/V) magnesium sulfate heptahydrate (manufactured by Nacalai Tesque,Inc.), an antifoamer, and water, was adjusted to 6.0. The liquid culturemedium was placed in ajar fermenter of 5 L capacity, followed byperforming autoclave treatment at 121° C. for 20 minutes to sterilizethe liquid culture medium. After the liquid culture medium was cooled,40 mL of the culture liquid disclosed in the above paragraph 1-1 (Seedcultivation) was seeded to the liquid culture medium, followed bycultivating fungus bodies for 41 hours under aerating and agitatingconditions. The culture liquid was filtered to obtain a culturesupernatant as a crude enzyme liquid 1.

1-3 (Obtaining Crude Enzyme Liquid by Solid Cultivation (BranCultivation))

300 g of wheat bran (manufactured by Yowa Seifun Co., Ltd.) and 240 g oftap water were put into a conical flask of 5 L capacity, and thenstirred well. The conical flask was plugged with cotton, and thensterilized at 121° C. for 25 minutes.

5 mL of the seed culture liquid disclosed in the above-paragraph 1-1(Seed cultivation) was seeded, and then left still at 26° C. forcultivation. After cultivation was performed for 2 weeks whileoccasionally stirring for aeration, cultivated fungus bodies attachingto the bran were subjected to extraction using 5 L of 20 mM potassiumphosphate buffer (pH 7.5), and then filtration to obtain a supernatantas a crude enzyme liquid 2.

1-4 (Obtaining Crude Enzyme Liquid by Solid Cultivation (OatmealCultivation))

300 g of oatmeal (manufactured by Snow Brand Milk Products Co., Ltd.)and 240 g of tap water were put into a conical flask of 5 L capacity,and then stirred well. The conical flask was plugged with cotton, andthen sterilized at 121° C. for 25 minutes.

5 mL of the seed culture liquid disclosed in the above-paragraph 1-1(Seed cultivation) was seeded, and then cultivated for four days at 25°C. by leaving it still while occasionally stirring for aeration. Thecultivated fungus bodies attaching to the oatmeal were subjected toextraction using 5 L of 20 mM potassium phosphate buffer (pH 7.5), andthen filtration to obtain a supernatant as a crude enzyme liquid 3.

1-5 (Purification of Enzyme)

The crude enzyme liquids 1 to 3 were subjected to enzyme purification bythe following steps (1) to (5) to isolate the coenzyme-linked glucosedehydrogenase.

(1) Concentration and Desalination

Each crude enzyme liquid was concentrated using an ultrafiltrationfilter “Pellicon 2 modules” (manufactured by Millipore Corporation) witha molecular weight cutoff of 10,000, and then substituted with 20 mMpotassium phosphate buffer (pH 7.5) to obtain each crude enzymeconcentrate.

(2) Purification Using Butyl-TOYOPEARL650M (Manufactured by TosohCorporation) (First Time)

The above-mentioned crude enzyme concentrate was adjusted to be a 65%ammonium sulfate saturated solution (pH 7.5), and then centrifuged toobtain a supernatant. The crude enzyme supernatant was passed through aButyl-TOYOPEARL650M column which was previously equilibrated with 20 mMpotassium phosphate buffer (pH 7.5) containing 65% saturated ammoniumsulfate so that the enzyme was adsorbed to the column. After the columnwas washed with the same buffer, the enzyme was eluted using 20 mMpotassium phosphate buffer (pH 7.5) containing 30% saturated ammoniumsulfate to collect an active fraction. Moreover, the enzyme was elutedby a gradient elution method using the same buffer to 20 mM potassiumphosphate buffer (pH 7.5), and then mixed with the above-mentionedactive fraction.

(3) Purification Using DEAE-CELLULOFINE A-500 (Manufactured by SEIKAGAKUCORPORATION)

The above-mentioned activity fraction was concentrated using anultrafiltration filter “Pellicon 2 modules” with a molecular weightcutoff of 10,000, and then desalinated, followed by equilibrating with15 mM Tris-hydrochloride buffer (pH 8.5). A DEAE-CELLULOFINE A-500column was equilibrated with the same buffer, and the the fraction waspassed through the column to collect an active fraction.

(4) Purification Using Butyl-TOYOPEARL650M (Manufactured by TosohCorporation) (Second Time)

The activity fraction was adjusted to be a 65% ammonium sulfatesaturated solution (pH 7.5), and then centrifuged to obtain asupernatant. The supernatant was passed through a Butyl-TOYOPEARL650Mcolumn previously equilibrated with 20 mM potassium phosphate buffer (pH7.5) containing 65% saturated ammonium sulfate so that the enzyme wasadsorbed to the column. After the column was washed with the samebuffer, the enzyme was eluted with 20 mM potassium phosphate buffer (pH7.5) containing 30% saturated ammonium sulfate to collect an activefraction.

(5) Purification Using TSK-Gel G3000SW (Manufactured by TosohCorporation)

The above-mentioned active fraction was concentrated with a pencil-typemembrane concentration module “ACP-0013” with a molecular weight cutoffof 13,000 (manufactured by Asahi Kasei Corporation), and thendesalinated, followed by equilibrating with 50 mM potassium phosphatebuffer (pH 5.5) containing 0.2 M sodium chloride. The fraction waspassed through a TSK-gel G3000SW (with a diameter of 2.15 cm and aheight of 60 cm) equilibrated with the same buffer, and then the enzymewas eluted with the same buffer to collect an active fraction. Theactive fraction was concentrated with CENTRIPLUS 10 (manufactured byAmicon Inc.), and then desalinated, followed by being substituted with50 mM sodium citrate/phosphate buffer (pH 5.5). The specific activity ofthe enzyme purified from the crude enzyme liquid 1 (hereinafter,referred to as “purified enzyme 1”) was approximately 1,800 units/mg.The specific activity of the enzyme purified from the crude enzymeliquid 2 (hereinafter, referred to as “purified enzyme 2”) wasapproximately 1,010 units/mg. The specific activity of the enzymepurified from the crude enzyme liquid 3 (hereinafter, referred to as“purified enzyme 3”) was equivalent to the above. The purification foldof each enzyme was 100-fold or more with respect to the crude enzymeliquid.

Example 2

(Preparation of Vector Containing Insert DNA)

(1) Isolation of Whole RNA

2 g of wet fungus bodies cultivated by the method described in the aboveparagraph 1-1 (Seed culture) of Example 1 were frozen with liquidnitrogen, and then 1.5 mg of whole RNA thereof was extracted using EASYPrep RNA (manufactured by TAKARA BIO INC.).

(2) Preparation of cDNA Library

A cDNA library was prepared from the whole RNA by performing reversetranscription using a reverse transcriptase and oligo dT adaptor primer.As a regent, “3′-Full RACE Core Set” (manufactured by TAKARA BIO INC.)was used under conditions disclosed in the protocol of the operatingmanual thereof.

(3) Cloning of GLD Gene

The GLD gene was amplified by PCR using the cDNA library as a template.As primers, plural oligonucleotides were synthesized based on theN-terminal and internal amino acid sequence of purified enzyme 2 freefrom embedding sugar, the amino acid sequence being determined byEdman's method, the purified enzyme 2 being obtained by purifying thecrude enzyme liquid 2 in accordance with the method described in theabove-paragraph 1-5 (purification of enzyme) of Example 1, and the crudeenzyme liquid 2 being obtained by bran cultivation as described in theparagraph 1-3 (solid cultivation (bran cultivation)) of Example 1.Finally, a primer set of KpnF (SEQ ID NO. 3) and PstR (SEQ ID NO. 4)primers was used to obtain an objective GLD gene.

PCR was performed using a DNA polymerase and Pyrobest (manufactured byTAKARA BIO INC.) by performing 25 cycles of (94° C. for 30 seconds→55°C. for one minute→72° C. for two minutes).

Then, a pColdII vector (manufactured by TAKARA BIO INC.) was cleavedwith restriction enzymes PstI and KpnI, and then the PCR-amplifiedfraction treated with the same restriction enzymes was ligated to thevector, followed by being transfected to an Escherichia coli bacteriumDH5α strain for transformation. Plasmid DNAs were prepared from 6 clonesof obtained transformants, and then treated with the restriction enzymesPstI and KpnI, as a result of which each clone was conformed to have afraction with an objective size. Among them, plasmids of 4 clones wereprepared to determine the sequence of the insert contained therein, andeach plasmid was confirmed to have an objective gene.

Example 3

(Transformation of Host and Purification of Enzyme)

A host Escherichia coli bacterium JM109 strain was transformed with therecombinant vector (pCGLD) prepared in Example 2, and transformant wasselected on LB agar medium containing ampicillin. Then, the transformantwas seeded in LB liquid culture medium containing 50 μg/ml ofampicillin, and then cultivated while shaking at 37° C. When the OD600of the cultivated liquid reached approximately 0.4 to 0.5, thecultivated liquid was cooled to 15° C., and then left still for 30minutes, followed by adding 1 mM IPTG thereto, and then furthercultivating while shaking at 15° C. for 24 hours. After the cultivationwas ended, the fungus bodies were collected by centrifugation, and thensuspended with 10 mM potassium phosphate buffer (pH 7.5). After thefungus bodies were sonicated using a sonicator, a cell-free extract wasobtained by centrifugation. It was confirmed by SDS-PAGE and activitymeasurement that an enzyme with an anticipated molecular weight wasexpressed. Also, it was confirmed that the enzyme activity was 0.09 U/mLof the cultivated liquid.

Moreover, the coenzyme-linked glucose dehydrogenase was isolated andpurified by the following steps (1) to (5).

(1) Concentration

The above-mentioned cell-free extract was concentrated with anultrafiltration filter “Pellicon 2 modules” with a molecular weightcutoff of 10,000 (manufactured by Millipore Corporation), followed bysubstituting with 20 mM potassium phosphate buffer (pH 7.5) to obtain acrude enzyme liquid.

(2) Purification Using Butyl-TOYOPEARL650M (Manufactured by TosohCorporation) (First Time)

The above-mentioned crude enzyme liquid was adjusted to be a 65%ammonium sulfate saturated solution (pH7.5), followed by centrifuging toobtain a supernatant. The obtained crude enzyme supernatant was passedthrough a Butyl-TOYOPEARL650M column previously equilibrated with 20 mMpotassium phosphate buffer (pH 7.5) containing 65% ammonium sulfate sothat the enzyme was adsorbed. After the column was washed with the samebuffer, the enzyme was eluted with 20 mM potassium phosphate buffer (pH7.5) containing 30% ammonium sulfate to collect an active fraction.Furthermore, the enzyme was eluted by a gradient elution method usingthe same buffer to 20 mM potassium phosphate buffer (pH 7.5), and thecollected active fraction was added to the above-mentioned activefraction.

(3) Purification Using DEAE-CELLULOFINE A-500 (Manufactured by SEIKAGAKUCORPORATION)

The above-mentioned active fraction was concentrated with anultrafiltration filter “Pellicon 2 modules” with a molecular weightcutoff of 10,000, and then desalinated, followed by equilibrating with15 mM Tris-hydrochloride buffer (pH8.5). The fraction was passed througha DEAE-CELLULOFINE A-500 column equilibrated with the same buffer tocollect the eluant.

(4) Purification Using Butyl-TOYOPEARL650M (Manufactured by TosohCorporation) (Second Time)

The eluant was adjusted to be a 65% ammonium sulfate saturated solution(pH 7.5), and then centrifuged to obtain a supernatant. The supernatantwas passed through a Butyl-TOYOPEARL650M column previously equilibratedwith 20 mM potassium phosphate buffer (pH 7.5) containing 65% ammoniumsulfate so that the enzyme was adsorbed. After the column was washedwith the same buffer, the enzyme was eluted with 20 mM potassiumphosphate buffer (pH 7.5) containing 30% ammonium sulfate to collect anactive fraction.

(5) Purification Using TSK-Gel G3000SW (Manufactured by TosohCorporation)

The above-mentioned active fraction was concentrated with a pencil-typemembrane concentration module “ACP-0013” (manufactured by Asahi KaseiCorporation.) with a molecular weight cutoff of 13,000, and thendesalinated, followed by equilibrating with 50 mM potassium phosphatebuffer (pH 5.5) containing 0.2 M sodium chloride. The fraction waspassed through TSK-gel G3000SW (with a diameter of 2.15 cm and a heightof 60 cm) equilibrated with the same buffer, and then the enzyme waseluted with the same buffer to collect an active fraction. The activefraction was concentrated with CENTRIPLUS 10 (manufactured by AmiconInc.), and then desalinated, followed by substituting with 50 mM sodiumcitrate/phosphate buffer (pH 5.5). The obtained enzyme (hereinafter,referred to as “purified enzyme 4”) had a specific activity ofapproximately 2,450 units/mg and a purification fold of approximately50-fold with respect to the crude enzyme liquid.

Example 4

(Transformation of Mold and Purification of Enzyme)

As a host, a strain of A. oryzae NS4 was used. As disclosed in KnownDocument 1 (Biosci. Biotech. Biochem., 61(8), 1367-1369, 1997), thisstrain was bred in 1997 (Heisei 9) at the National Research Institute ofBrewing, has been used for analyzing transcription factors, culturingvarious strains with a high ability of yielding an enzyme, or the like,and has been commercially available.

A vector which can realize expression of the GLD gene was prepared usingan improved amylase promoter derived from A. oryzae for gene expressionin the strain as disclosed in Known Document 2 (Heterologous geneexpression system in the genus Aspergillus, Toshitaka Minetoki,Chemistry & Biology, 38, 12, P831-838, 2000).

Transformation was performed basically in accordance with a methoddisclosed in Known Document 2 and Known Document 3 (Genetic engineeringof Aspergillus oryzae for Japanese Sake, Masaya Gomi, Journal of theBrewing Society of Japan, pages 494 to 502, 2000). Selection oftransformant with activity was repeatedly performed to obtain a strainof Aspergillus oryzae with an ability of yielding the GLD.

The strain was cultivated while shaking at 30° C. for 5 days in a liquidculture medium containing 1% peptone, 2% sucrose, 0.5% dipotassiumhydrogen phosphate, and 0.05% magnesium sulfate to obtain a cultivatedliquid with a GLD activity.

Purification was performed in accordance with the same method as that ofExample 3, and then SDS polyacrylamide gel electrophoresis was performedto obtain an approximately single enzyme sample. The sample is referredto as “purified enzyme 5”.

Example 5

(Transformation of Yeast and Purification of Enzyme)

A host used was a strain prepared by improving a strain of Candidaboidinii S2 AOU-1, which is known as a yeast with a high ability ofyielding protein, in accordance with a method disclosed in KnownDocument 4 (Laboratory Manual for Gene Expression, Production of usefulprotein in high expression system, edited by Isao Ishida and Tamie Ando,Kodansya Scientific Ltd., pages 100 to 129, 1994) for transfection of aheterologous gene. The strain of S2 AOU-1 was named as Candida boidiniiSAM1958 and deposited in the National Institute of Bioscience andHuman-Technology as Accession No. FERM BP-3766 on Feb. 25, 1992.

A vector which realizes expression of the GLD gene in the improvedstrain was prepared using a promoter induced by methanol, the promoterbeing derived from the strain of S2 AOU-1 as disclosed in Known Document5 (Heterologous gene expression system by methanol-utilizing yeast,Hiroya Yurimoto and Yasuyoshi Sakai, Chemistry & Biology, 38, 8,P533-540, 2000). Then, transformants were selected in accordance withKnown Documents 4 and 5 to obtain a strain of Candida boidinii with anability of yielding the GLD.

The strain was cultivated while shaking at 28° C. for two days in aliquid culture medium containing 2% peptone, 1% yeast extract, 2%glycerol, and 1% methanol to obtain a cultivated liquid with a GLDactivity.

Purification was performed in accordance with the same method as that ofExample 3, and then SDS polyacrylamide gel electrophoresis was performedto obtain an approximately single enzyme sample. The sample is referredto as “purified enzyme 6”.

Example 6

(Enzymatic Decomposition)

0.1% (v/v) SUMIZYME PX and 0.1% (v/v) SUMIZYME ARS were added to aportion of the crude enzyme liquid 1 prepared in paragraph 1-2 ofExample 1 (obtaining crude enzyme liquid by liquid cultivation), andthen reacted at 40° C. for two hours. After the reaction was ended, thereactant was subjected to SDS-PAGE, and then sugar chain staining usinga sugar chain staining kit (Gelcode Glycoprotein Staining Kit(manufactured by PIERCE)) in accordance with a determined method.However, no decomposition of sugar chains could be recognized.

(Oxidation, Reduction, and Acid Hydrolysis by Metaperiodic Acid)

The crude enzyme liquid 1 was put into a 1.5 ml Eppendorf tube coveredwith aluminum foil in a protein amount of 20 μg, and then an eighth partof 0.8N sodium metaperiodate (NaIO₄) aqueous solution (finalconcentration thereof was 0.1N) was added thereto, followed byperforming oxidation at 25° C. for 24 hours. Then, to the solution, atenth part of 0.4N NaBH₄ aqueous solution was added, followed byperforming reduction at room temperature for 10 hours. Furthermore, tothis solution, a tenth part of 1N sulfuric acid aqueous solution wasadded, followed by performing hydrolysis at 25° C. for 24 hours. In thesame manner as that of the above-mentioned enzymatic decomposition test,the reactant was subjected to SDS-PAGE and then sugar chain stainingusing a sugar chain staining kit. However, no decomposition of sugarchains could be recognized.

(Electrophoresis (Sugar Chain Staining, Coomassie Brilliant Blue (CBB)Staining, and Activity Staining))

To each enzyme liquid (purified enzymes 1, 2, 4, 5, and 6) prepared inExamples 1 to 5, one unit of glycopeptidase F (manufactured by Wako PureChemical Industries, Ltd.) was added per 0.1 mg of a protein therein,and then reacted at 37° C. for 15 hours to cut sugar chains, followed bysubjecting to SDS-PAGE. The SDS-PAGE gel was subjected to sugar chainstaining to confirm the amount of sugar chains and whether the sugarchains were cut. The sugar chain staining was performed using a sugarchain staining kit (Gelcode Glycoprotein Staining Kit (manufactured byPIERCE)) in accordance with a determined method (FIG. 1). As a result,existence of a great deal of sugar in the purified enzyme 1 preparedfrom the liquid cultivation supernatant was confirmed, and no differencebetween before and after treatment of cutting sugar chains wasrecognized, and therefore, it was assumed that the glycopeptidase Fprovided no influence thereon. On the other hand, it was confirmed thatthe molecular weights of the purified enzymes 2, 5, and 6, purified fromthe products cultivated in a solid state, decreased by cutting the sugarchains, and a portion of the sugar chains was cut with theglycopeptidase F.

Also, when SDS-PAGE and then Coomassie Brilliant Blue (CBB) stainingwere performed in the same way, the purified enzyme 1 was hardly stainedwith CBB, but the purified enzymes 2, 4, 5, and 6 were stained well withCBB (FIG. 2). As is apparent from the above results, the purifiedenzymes prepared by solid cultivation or gene recombination had a lowerbinding sugar content and were easily stained with CBB in comparisonwith the purified enzyme prepared by liquid cultivation.

Also, electrophoresis analysis was performed by native-PAGE using anelectrophoresis gel of NPU-7.5L for native-PAGE (manufactured by ATTOCORPORATION), and then an activity staining was performed. Resultsthereof are shown in FIG. 3. Lane 7 indicates a result of a sampleprepared by liquid cultivation, the enzyme activity thereof beingadjusted to 20 mU. Lane 8 indicates a result of a sample prepared bybran cultivation, the enzyme activity thereof being adjusted to 20 mU.Lane 9 indicates a result of a sample prepared by oatmeal cultivation,the enzyme activity thereof being adjusted to 20 mU. Lane 7 indicates aresult of the purified enzyme 1, lane 8 indicates a result of thepurified enzyme 2, and lane 9 indicates a result of the purified enzyme3.

The electrophoresis position of the purified enzymes 2 and 3, which arepurified from the products cultivated in a solid state, was lower thanthat of the purified enzyme 1 purified from the supernatant cultivatedin a liquid state. As is apparent from the above results, the sugarcontent in the obtained enzyme was decreased by changing the cultivationstate from a liquid state to a solid state, and thereby, the obtainedenzyme was easily stained with CBB.

In consideration of the results of the above examples, it was assumedthat the purified enzyme 1 and the other purified enzymes differed inthe sugar content and sugar composition, and so the sugar analysis ofthe enzymes was performed.

Example 7

(Analysis of Sugar Composition by ABME Labeling—HPLC Analysis)

First, 35 mg of methyl p-aminobenzoate (ABME) and 3.5 mg of sodiumcyanoborohydride were put into a test tube, and then 350 μl of methanoland 41 μl of acetic acid were added, followed by stirring the mixture.

Each purified enzyme liquid prepared in the above-mentioned Examples 1to 5 was adjusted to have a protein content of 1.0 mg/ml, and then 100μl thereof was put into a test tube equipped with a screw cap, followedby drying up to harden under a nitrogen gas stream, adding 0.2 ml of 4NTFA (trifluoroacetic acid) solution thereto, and then reacting at 100°C. for 4 hours. After the reaction was ended, the reactant was dried upto harden under a reduced pressure in a tube evaporator, followed byadding 200 μl of ion-exchanged water thereto. Then, the drying-upprocedure was repeated three times, and thus TFA was completely removed.In a fume hood, 200 μl of methanol, 20 μl of pyridine, and 20 μl ofacetic acid anhydride were added to the resultant, and then left stillat room temperature for two hours or longer to perform N-acetylation.The reactant liquid was dried up to harden under a nitrogen gas stream,and then dissolved in 1 ml of ion-exchanged water, followed by passingit through a cartridge column of PRE-SEP C18 (manufactured by WatersCorporation) previously washed, and then performing elution with 15 mlof ion-exchanged water. The eluant was concentrated under a reducedpressure using a rotary evaporator, and then transferred to a test tubeequipped with a screw cap, followed by drying up to harden in a tubeevaporator. The residue was dissolved in 20 μl of ion-exchanged water,and then 80 μl of the ABME reagent was added thereto, followed byreacting at 80° C. for 45 minutes. After the reaction was ended, thereactant liquid was dried up to harden under a nitrogen gas stream,followed by adding 2 ml of ion-exchanged water and 2 ml of diethyl etherthereto, and then stirring. The resultant was centrifuged and an etherlayer containing unreacted ABME was removed. This ether extraction wasrepeated five times, and the obtained aqueous layer was dried up toharden in a tube evaporator to obtain a saccharide derivatized withABME. This saccaride was dissolved in 2 ml of high-purity water toperform HPLC analysis.

A column used for HPLC analysis was Wakosil 5C18-200 (4.0×250 mm;manufactured by Wako Pure Chemical Industries, Ltd.), the columntemperature was 40° C., the flowing rate was 0.5 mL/min, and solventsused were a mixture composed of 5% acetonitrile and 0.1M acetic acidsolution (solvent A), and a mixture composed of 15% acetonitrile and0.1M acetic acid solution (solvent B). Elution was performed in asolvent ratio A:B of 100:0 for 20 minutes after the sample was injected,and then elution was performed in such a linear solvent's concentrationgradient manner that the solvent ratio A:B was changed to 0:100 over 80minutes. UV detection wavelength was 304 nm.

As a result, galactose, mannose, arabinose, rhamnose, andN-acetylglucosamine were detected from the purified enzyme 1 derivedfrom the product cultivated in a liquid state, but glucose was notdetected therefrom. On the other hand, mannose and N-acetylglucosaminewere detected from the purified enzyme 2 derived from the productcultivated with bran, but glucose, galactose, arabinose, and rhamnosewere not detected therefrom. In addition, the sugar content of eachpurified enzyme is shown in Table 1.

TABLE 1 Galactose Glucose Mannose Arabinose Xylose RhamnoseN-acetylglucosamine Purified Enzyme 1 11.9 ND 1.72 29.0 ND 1.72 0.434(derived from liquid-cultivated wild strain) Purified Enzyme 2 ND ND0.269 ND ND ND 0.045 (derived from bran-cultivated wild strain) PurifiedEnzyme 5   0.046 ND 0.072 ND MD ND 0.015 (derived from recombinant mold)Purified Enzyme 6 ND 0.273 0.644 ND ND ND 0.023 (derived fromrecombinant yeast)

Example 8

(Quality Examination of Coenzyme-Linked Glucose Dehydrogenase)

The purified enzymes 1 to 6, isolated in the above-mentioned Examples 1to 5, were examined in terms of activity, substrate selectivity,inhibitor and coenzyme thereof.

The enzyme activity was measured in accordance with methods disclosed asEnzyme activity measurement method-1 and Enzyme activity measurementmethod-2 on pages 36 and 37 of the specification of WO2004/058958.

1) Activity

Each purified enzyme was reacted with 500 mM D-glucose in the presenceof 8.66 mM DCIP, and the resultant was subjected to quantitativeanalysis using a kit for D-gluconic acid/D-glucono-δ-lactonequantitative analysis (manufactured by Roche Diagnostics K.K.). As aresult, it was confirmed that D-gluconic acid was produced in eachpurified enzyme, and thus it was revealed that the purified enzymes 2 to6 were also enzymes which catalyze a reaction in which a hydroxyl groupat the 1-position of D-glucose was oxidized in the same way as thepurified enzyme 1.

2) Optimum pH:

The enzyme activity of the purified enzyme at various pH regions wasmeasured in a similar way to Enzyme activity measurement method 2 exceptthat a potassium phosphate buffer (pH 6.0 to 7.0), Tris-hydrochloridebuffer (pH 7.4 to 8.0), or glycine-sodium hydroxide buffer (pH 8.6 to9.1) (each buffer being used at the final concentration of 17 mM) wassuitably used instead of a buffer used in the measurement method 2. As aresult, the optimum pH of the purified enzymes 4, 5, and 6 was 7.0 to9.0.

3) Stable pH

The purified enzyme was dissolved in 50 mM of each buffer, that is,acetic acid/sodium acetate buffer (pH 3.6 to 5.3), potassium phosphatebuffer (pH 6.0 to 6.8), Tris-hydrochloride buffer (pH 7.7), orglycine-sodium hydroxide buffer (pH 8.6 to 10.0), and then held at 40°C. for 60 minutes, followed by measuring the enzyme activity inaccordance with the activity measurement method-1 to analyze theresidual rate of the enzyme activity. The stable pH of the purifiedenzyme 5 was 4.5 to 8.5.

4) Optimum Temperature

The coenzyme-linked glucose dehydrogenase was dissolved in 50 mMpotassium phosphate buffer (pH 7.0), and then the enzyme activity in thetemperature region between 30° C. and 62° C. was measured in accordancewith the activity measurement method-1. As a result, the optimumtemperature of the purified enzyme 5 was approximately 55° C.

5) Temperature Stability

The coenzyme-linked glucose dehydrogenase was dissolved in 50 mMpotassium phosphate buffer (pH 7.0), and then held for 15 minutes at atemperature within the range of 0° C. to 55° C., followed by measuringthe enzyme activity in accordance with the activity measurement method-1to analyze the residual rate of the enzyme activity. The residual rateof the enzyme activity was calculated with respect to the enzymeactivity exhibited when held at 0° C. for 15 minutes which is assumed tobe 100%. As a result, 89% of the enzyme activity of the purified enzyme5 was sustained even at 50° C., and the enzyme activity was stable atapproximately 50° C. or lower.

6) Subunit Molecular Weight:

The purified enzyme was subjected to SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) using 12.5% polyacrylamide gel in accordancewith a method disclosed by Laemmli et al., (Nature (1970) 227: 680-685).After the electrophoresis was ended, the gel was stained with CoomassieBrilliant Blue (CBB), and the mobility of the enzyme was compared withthat of a molecular weight marker (LMW Marker; manufactured by AmershamPharmacia Biotech, Inc.), as a result of which it was revealed that thesubunit molecular weight of each enzyme was as follows: the subunitmolecular weight of the purified enzyme 2 was approximately 71 kDa, thatof the purified enzyme 4 was approximately 58 kDa, that of the purifiedenzyme 5 was approximately 81 kDa, and that of the purified enzyme 6 wasapproximately 128 kDa.

7) Substrate Selectivity

The enzyme activity of the purified enzymes 1 to 6 was measured in asimilar way to the enzyme activity measurement method-1 except thatD-glucose or each other substrate (each being used at the finalconcentration of 333 mM, excepting D-cellobiose being used at the finalconcentration of 193 mM, and D-trehalose and D-raffinose being used atthe final concentration of 121 mM) was used instead of the substrate inthe reaction liquid for measuring the activity by the activitymeasurement method-1. The activity against each substrate was calculatedas a relative value with respect to the activity against D-glucose whichis assumed to be 100%.

The enzyme activity was measured in a similar way to the above exceptthat D-glucose was used at the final concentrations of 550 mM and 100 mMand maltose was used at the final concentrations of 550 mM and 100 mM,and then the relative activity (enzyme activity) thereof was determined.The activity toward maltose was calculated as a relative value withrespect to the activity toward D-glucose.

In the same way as that of the purified enzyme 1, the purified enzymes 2to 6 according to the present invention strongly acted on D-glucose, butweakly acted on D-mannose, 1,5-anhydro-D-glucitol, D-cellobiose,D-trehalose, maltose, D-galactose, D-glucose-6-phosphate, andD-fructose. The purified enzymes 2 to 6 provided almost no action onL-arabinose, lactose, D-sorbitol, gluconic acid, sucrose, D-mannitol,L-sorbose, D-ribose, L-rhamnose, D-glucose-1-phosphate, D-raffinose,ethanol, and glycerol.

8) Inhibitor

To the reaction system in the activity measurement method-1,1,10-phenanthroline dissolved in methanol so that each finalconcentration was 1 mM, 5 mM, 10 mM, 25 mM, or 50 mM, was added,followed by measuring the activity of the purified enzymes 1 to 6 by theactivity measurement method-1. Each final concentration of methanol inthe reaction system was 10% (v/v). As a control, methanol was added tothe reaction system in the activity measurement method-1 at the finalconcentration of 10% (v/v), and then the activity was measured by theactivity measurement method-1. As a result, each inhibition ratiorealized by 1,10-phenanthroline formulated at the final concentration of1 mM or more was 60% or more, which was high.

9) Coenzyme

D-glucose was added to the purified enzymes 1 to 6, and then absorptionspectrometry was performed. In each case, the absorption maximumsrecognized at 385 nm and 465 nm disappeared by adding D-glucose, andthus it was revealed that a coenzyme thereof was FAD. The absorptionmaximums are specific to FAD, and cannot be recognized in a controlreaction system in which only FAD is not contained.

Example 9

(Comparison of Sensor Characteristics)

Each bimolecular reaction rate constant of the purified enzyme 1prepared in the paragraph 1-2 of Example 1 and the purified enzymes 4 to6 prepared in Examples 3 to 5 was determined using an electrochemicalanalyzer of CHI611A (manufactured by BAS Inc.). A platinum auxiliaryelectrode, a carbon work electrode, and a silver/silver chloridereference electrode were used. To MOPS buffer with pH of 7.0, 142 mM ofglucose, and any one of 0.45 μM of the purified enzyme 4, 0.76 μM of thepurified enzyme 5, 1.9 μM of the purified enzyme 6, and 1.1 μM of thepurified enzyme 1 were added, each concentration indicating the finalconcentration, followed by adding an osmium complex[Os(4-methyl-imidazole)₂(4-dimethyl-bipyridine)₂](PF₆)₂ at the finalconcentration of 0 mM to 0.57 mM, and then recording a cyclicvoltammogram at each concentration (see FIG. 4). As a result, it wasrevealed that the bimolecular reaction rate constant of the purifiedenzyme 4 was 8.15×10⁴ s⁻¹M⁻¹, the bimolecular reaction rate constant ofthe purified enzyme 5 was 7.36×10⁴ s⁻¹M⁻¹, and the bimolecular reactionrate constant of the purified enzyme 6 was 9.38×10⁴ s⁻¹M⁻¹. The purifiedenzyme 1 provided so low a current that the steady current value couldnot be found, and therefore, the bimolecular reaction rate constantcould not be calculated (FIG. 4).

In the same way, 142 mM of glucose, and any one of 0.45 μM of thepurified enzyme 4, 0.76 μM of the purified enzyme 5, 0.55 μM of thepurified enzyme 6, and 1.1 μM of the purified enzyme 1 were added toMOPS buffer with pH of 7.0, each concentration indicating the finalconcentration, followed by adding a quinone compound,2,3-dimethoxy-5-methyl-1,4-benzoquinone, at the final concentration of 0mM to 0.22 mM, and then recording cyclic voltammogram at eachconcentration (see FIG. 5). As a result, it was revealed that thebimolecular reaction rate constant of the purified enzyme 4 was 1.14×10⁸s⁻¹M⁻¹, the bimolecular reaction rate constant of the purified enzyme 5was 5.29×10⁷ s⁻¹M⁻¹, and the bimolecular reaction rate constant of thepurified enzyme 6 was 2.49×10⁷ s⁻¹M⁻¹. The purified enzyme 1 provided alow bimolecular reaction rate constant of 5.69×10⁴ s⁻¹M⁻¹.

The results show that the enzymes derived from genetically engineeredcells exhibited improved reactivity in comparison with the enzymederived from a wild strain.

Each bimolecular reaction rate constant of the purified enzymes 1 and 2prepared in the paragraphs 1-2 and 1-3 of Example 1 was determined usingan electrochemical analyzer of CHI611A (manufactured by BAS Inc.). Aplatinum auxiliary electrode, a carbon work electrode, and asilver/silver chloride reference electrode were used. 142 mM of glucoseand either 0.94 μM of the purified enzyme 1 or 3.3 μM of the purifiedenzyme 2 were added to MOPS buffer with pH of 7.0, each concentrationindicating the final concentration, followed by adding potassiumferricyanide at the final concentration of 0 mM to 0.671 mM, and thenrecording a cyclic voltammogram at each potassium ferricyanideconcentration (0, 0.019, 0.048, 0.095, 0.142, 0.188, 0.234, 0.280,0.325, 0.370, 0.414, 0.458, 0.501, 0.544, 0.587, 0.629, and 0.671 mM).As a result, it was revealed that the bimolecular reaction rate constantof the purified enzyme 2 was 2.84×10³ s⁻¹M⁻¹. The purified enzyme 1provided so low a current that the steady current value could not befound, and therefore, the bimolecular reaction rate constant could notbe calculated. It was assumed that the purified enzyme 1 exhibited lowreactivity because it was a sugar-embedded type enzyme, while thepurified enzyme 2 exhibited improved reactivity because it was an enzymewith normal sugar chains.

Example 10

(Measurement of Glucose Using Enzyme-Immobilized Electrode)

The concentration of D-glucose was measured by an enzyme-immobilizedelectrode using each purified enzymes 1, 2, 4, 5, and 6. The currentvalue in response to the glucose concentration was measured using aglassy carbon (GC) electrode in which 1.0 U of each enzyme wasimmobilized. 1.8 ml of 50 mM sodium phosphate buffer (pH 7.0) and 0.2 mlof 1M potassium hexacyanoferrate (III) (potassium ferricyanide) aqueoussolution were put into an electrolysis cell. After the GC electrode wasconnected to a potentiostat of BAS100B/W (manufactured by BAS), thesolution was stirred at 40° C. and the voltage of +550 mV was applied toa silver/silver chloride reference electrode. To this system, 20 μl of1M D-glucose solution was added and the current value at thesteady-state was measured. In addition, the procedure in which the sameamount of 1M D-glucose solution was added and then the current value wasmeasured was repeated three times. The measured current values wereplotted against the known glucose concentrations (approximately 10, 20,30, and 40 mM) to generate each standard curve (FIG. 6). Thus, it wasdemonstrated that quantitative analysis of glucose can be realized bythe enzyme-immobilized electrode using the OLD according to the presentinvention.

Example 11

Plural oligonucleotides were synthesized based on the sequenceinformation set forth in SEQ ID NO. 1, and then oligonucleotides setforth in SEQ ID NO. 13 and SEQ ID NO. 14 were finally selected as aprimer set. PCR was performed using the primer set and a template DNAderived from each strain with an ability of yielding the coenzyme-linkedglucose dehydrogenase of which a coenzyme is FAD, that is, Aspergillusjaponicus IFO4408, Penicillium cyaneum IFO5337, and Ganoderma applanatumIFO6498. The template DNA was prepared by cultivating each strain inaccordance with the method described in Example 1 to obtain wet fungusbodies, freezing the wet fungus bodies with liquid nitrogen, crushingthe fungus bodies, and then extracting with a mixture ofphenol/chloroform/isoamyl alcohol (25:24:1) (manufactured by NIPPON GENECO., LTD.). PCR was performed in 35 cycles of (94° C. for 30 seconds,42° C. for 30 seconds, and then 72° C. for 1.5 minutes) using TaKaRa LATaq (manufactured by TAKARA BIO INC.) and a thermal cycler (manufacturedby Stratagene Corp.). Each sequence of amplified products with a lengthof approximately 1.6 kbp was analyzed. The cDNA sequence free fromintrons was compared with the sequence set forth in SEQ ID NO. 1 andsequences of a known glucose oxidase and sorbose dehydrogenase. As aresult, it was revealed that nucleotide sequences (set forth in SEQ IDNOs. 5 to 7) and amino acid sequences (set forth in SEQ ID NOs. 8 to 12)are specific to the coenzyme-linked glucose dehydrogenase of which acoenzyme is FAD. In particular, it is assumed that the amino acidsequence set forth in SEQ ID NO. 8 is a binding site of FAD and is aportion of the active center.

INDUSTRIAL APPLICABILITY

The present invention can be utilized in a field of examination ofdiabetes.

The invention claimed is:
 1. A method for producing a recombinantglucose dehydrogenase (GLD) comprising: (i) transforming a fungal oryeast host cell with a recombinant expression vector comprising anucleic acid encoding a GLD comprising the sequences set forth in SEQ IDNOs. 8 to 12, wherein in SEQ ID NO:8, Xaa at residue #2 is A, and/or Xaaat residue #8 is V, and/or Xaa at residue #9 is I; wherein in SEQ IDNO:10, Xaa at residue #3 is L, and/or Xaa at residue #4 is L, and/or Xaaat residue #10 is V; and wherein in SEQ ID NO:12, Xaa at residue #5 isS, and/or Xaa at residue #8 is N, and/or Xaa at residue #9 is I; (ii)culturing the transformed host cell and recombinantly expressing theGLD; and (iii) collecting recombinant GLD secreted from the transformedhost cell.
 2. The method of claim 1, wherein the recombinant GLD has anactivity toward maltose of 5% or less with respect to its activitytoward glucose.
 3. The method of claim 1, wherein the recombinant GLDhas a total content of galactose, glucose, mannose, and arabinose of 10μg or less per μg of protein.
 4. The method of claim 1, wherein therecombinant GLD has a total content of galactose, glucose, mannose, andarabinose of 2 μg or less per μg of protein.
 5. The method of claim 1,wherein the host cell is a fungal cell.
 6. The method of claim 1,wherein the host cell is a yeast cell.
 7. The method of claim 5, whereinthe collecting of the recombinant GLD comprises isolating therecombinant GLD from culture medium containing the recombinant GLDsecreted from the fungal host cell.
 8. The method of claim 1, whereinthe recombinant GLD is an Aspergillus species GLD.
 9. The method ofclaim 8, wherein the recombinant GLD is an Aspergillus oryzae speciesGLD.
 10. The method of claim 9, wherein SEQ ID NO:9 isRAGKALGGTSTINGMAYTRAEDVQID.